JPH05504060A - Coexisting binding angiogenin/RNase hybrid - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Background of the invention of co-associated angiogenin/RNase hybrid 1 Field of invention The present invention provides modified or mutated angiogenin proteins and modified or mutated proteins. Concerning the encoding DNA sequence. Additionally, the present invention provides modified or mutant angiogens. This article relates to vectors, host cells, and expression methods for expressing protein proteins. Especially books The modified or mutant angiogenin-inducing proteins of the invention are genetically engineered covalently linked angiogenin-inducing proteins. It is an ogenin/ribonuclease (RNase) hybrid. this hive The lid is a structural component of angiogenin that is necessary for its characteristic activity. This is useful for identifying the number of minutes. In particular, local mutagenesis allows special of angiogenin which is partially replaced by the corresponding RNase segment. to produce angiogenin/RNase hybrid-proteins that are derivatives of It can be used to The production and characterization of such proteins is an Segments, such as the N-terminal segment according to the present application, are critical to the characteristic activity of ogenin. ment identification. In particular, the present application describes the N-terminal residue of angiogenin 8-2. 2 by the corresponding portion of the RNase (residues 7-21) by local mutagenesis. A novel recombinant hybrid protein with the ability to promote angiogenesis, ARH- Regarding m. Such recombinant mutant angiogenin protein is useful for its clinical treatment. can be manufactured in sufficient quality to permit its administration as a drug or diagnostic agent .

2 Technology background Induction of angiogenesis, the process of growing a vascular network, is essential for solid tumor growth. and is a component of normal wound healing and growth processes. This is atherogenesis, joint There is also a deep connection with the pathophysiology of inflammation and diabetic retinopathy. This is a specific stimulus characterized by controlled growth of new capillaries. This growth is within It appears to be mediated by epithelial cell migration and proceeds independently of endothelial cell mitosis. .

The molecule responsible for the process of angiogenesis induction has already been long sought. came. Greenblatt and Shubik (5hub) ik) in 1968, J. Natl. Cancer In5t, Vol. 41, On pages 112-124, tumor-induced neotube formation is mediated by diffusible substances. I inferred that it would. Subsequently, a variety of soluble mediators play a role in inducing neofacial tube formation. associated. Prostaglandin (author: ^uerbach, Lyspok- ines, Pfck and Landy Publishing, pp. 69-88, AcademicP. ress, New York, 1981), human urokinase (Berm an et al., 1982, Invest, Optala, Vis, Sci.

Volume 22, pp. 191-199), Copper (Rajn et al., 1982, J, Na tl, Cancer In5t, Vol. 69, pp. 1183-1188), and These include various "angiogenesis-inducing factors."

Many angiogenesis-inducing factors are present in tumor cells, wound fluid (Banda et al., 1982, P. roc, Natl, Acad, Sci, USA, Volume 79, No. 7773 ~7777 pages; Banda et al., U.S. Pat. No. 4,503,038) and Web Membrane cells (D'^ Cao ore, 1981, Proc, Natl, Acad, S ci, USA, Vol. 78, pp. 3068-3072).

Tumor-induced angiogenesis inducers are generally poorly characterized. Volkmann (1'01kian) et al. (1971, J, Exp, 1led, vol. 133 Walker 2 It was isolated from 56 rat ascites tumors. This factor promotes division of capillary endothelial cells. aggressive and inactivated by RNase. Tuan et al. (1 973, Biochemistry, Vol. 12, pp. 3159-3165) Mitogenic and angiogenic activities found in non-histone proteins of worker-256 tumors. I started it. This active fraction was a mixture of proteins and carbohydrates. many demonstrated that animal and human tumors produce angiogenesis-inducing factors (Ph111i ps and 1unar, 1979, Int, J, Cancer, Volume 23 82-88), but the chemical properties of these factors have not been determined. worker 256 Low molecular weight non-protein components from tumors are also angiogenic and mitogenic. (Weiss et al., 1979, Br. J. Cancer , Vol. 40, pp. 493-496). Blood with a molecular weight of 400-800 daltons Although the tube formation-inducing factor was purified to homogeneity (Fengelau et al., 1981; J, Biol, Chew, Vol. 256, pp. 9605-9611), further features I couldn't attach it.

Human lung tumor cells are composed of polymeric carriers and low molecular weight, possibly non-proteinaceous, active ingredients. (Kunar et al., 1983). Year, Int.

J, Cancer Vol. 32, Nos. 461-464). Vallee ) et al. (1985, Experientia, Vol. 41, pp. 1-15) Formation-inducing activity is associated with three fractions from worker-256 tumors. I discovered that. Tolbert et al. (U.S. Pat. No. 422,953) 1) discovered the production of angiogenesis-inducing factors from an incoming cancer cell line, HT-29. However, this substance has only been partially purified and has not been chemically characterized. borrowed. Isolation of the gene responsible for the production of the angiogenesis-inducing factor is essential to the purity of the factor. and even partially unreported due to lack of characterization.

Isolation of angiogenesis-inducing factors can be achieved by applying many different techniques, such as high-performance liquid chromatography. Matography (Banda et al., supra), solvent extraction (Folk an et al., supra). ), chromatography on silica gel (Fen5elau et al., supra), DE AE cell o-su (1eiss et al., supra), and f;! −t=77 decks (Tuan et al., supra), and affinity chromatography (Teiss et al., supra). ).

(see U.S. Pat. No. 4,727,137) developed blood vessels from incoming cancer cell lines. The formation-induced protein was purified. This protein was confirmed in normal human plasma (5 h a-piro et al., 1987, Bfochem, Vol. 26, No. 5141-51 (page 46). The purified protein known as angiogenin has not been chemically characterized. and its amino acid sequence has been determined. Two different activities induce human tumor induction. Proven for Ogenin. First, it is a very strong angiogenesis-inducing factor in vivo. (Fett et al., 1985, Bioc hem, Vol. 24, pp. 5480-5486). Second, two points are clear. A characteristic enzyme for 28S and 18S rRNA that is different from crab pancreatic RNase. It was found that: (a) r to the same extent as RNase; Angiogenin is required up to 105 to obtain RNA degradation: and (b) The product is larger, i.e. 100-500 nucleotides; is essentially inactive against primarily standard RNase A molecules. (5hapi ro et al., 1986, Biochem, Vol. 25, pp. 3527-3532; St, cl-air et al., 1987, Proc, Natl, ^cad, Sci, USA 1 Vol. 84, pp. 8330-8334).

Furthermore, Vuary et al. (U.S. Pat. No. 4,721,672, attached by reference) US Patent No. 4727 from liver cDNA library and human genome library The gene (cDN) encoding the angiogenesis-inducing protein claimed in No. 137 A and genome) were cloned. Angiogenin gene in vector The recombinant vector encoding the angiogenin gene is cloned into host cells. used for transformation or transfection. transformed in this way The transfected cells express human angiogenin protein.

Angiogenin as described and claimed in U.S. Pat. No. 4,721,672 Several groups have produced synthetic angiogenin genes based on the gene sequence. did. Denefle et al. (1987, Gene.

Volume 56, pp. 61-70) produced a synthetic gene encoding human angiogenin. However, this does not use the codons found in highly expressed E. coli. It is for the purpose of This synthetic gene was promoted to E. coli tryptophan (trp). ligated into a pBR322-inducible expression vector constructed to contain the Ta.

This E. coli-produced angiogenin is insoluble but easily renatured and purified. . Purified angiogenin was extracted from incoming cancer cells by Vuary et al. (U.S. Pat. No. 4,727). No. 137) describes purified natural angiogenin. showed similar angiogenic activity and ribonucleolytic acid degrading activity. Regarding angiogenin The synthetic gene for the pond is published by Hoechst (German patent application P371672). 2.7), which is disclosed in U.S. Pat. No. 4,721,672. found in the described and claimed natural (i.e. wild type) angiogenin gene. Instead of the excreted methionine, leucine is encoded at amino acid position 30.

This synthetic Leu-30 angiogenin gene is advantageously expressed in E. coli. It was intended for use with This gene has a modified trp promoter (European Patent No. 0198415) and translation initiation region (TIR) arrangement. (Gene, Vol. 41, pp. 201-206, 1986; EMBOJ, No. 4 Vol. 519-526, 1985). Increased translation efficiency. Thus, the synthetic gene directly controls the trp promoter. and expression is controlled by addition of indole-3-acrylic acid or by addition of indole-3-acrylic acid. Induced by liptophan deficiency. This Leu-30 angiogenin protein can be purified and similar to wild-type (let-30) angiogenin. It was found that it exhibits angiogenesis-inducing activity and ribonucleic acid salt degrading activity.

Whether plasma-derived or tumor cell-derived, recombinant DNA-derived (cDNA, genomic All human angiogenin proteins described, whether NA or synthetic NA), Exhibits both angiogenic and ribonucleolytic activity. Indeed, angiogeni One of its most interesting properties is its structural homology with mammalian pancreatic RNase. Ru. This structural relationship not only allows investigation of the mechanism of action of angiogenin. First, the angiogenesis-inducing activity and enzymatic (i.e. liponucleolytic acid degrading) activity of angiogenin. It also makes it possible to investigate relationships between the sexes.

In vivo, angiogenin is measured in the chicken chorioallantoic membrane (CAM). It is a powerful vascular growth inducer (Fett et al., 1985, Bf ochem-1stry, Vol. 24, pp. 5480-5486).

In vitro, angiogenin stimulates phospholipase C activation and prostaglandin. It induces a wide variety of responses in endothelial cells, including the secretion of cyclins (Bic Kn-ell and VBLLee, 1988, Proc, Natl, Acad. , Sci.

[ISA, Vol. 85, pp. 5961-5965; Bicknell and Val lee, 1989, Proc, Natl, cad, Sci.

υS^ Volume 86, pages 1573-1577): This is a 40S liposome subunit. The specific cleavage of 183 RNA in St. , Claire et al., 1987, Proc, Natl, Acad, Sc. i, US^, Volume 84, Pages 8330-8334: St, Claire et al. , 1988, Biochemistry, Vol. 27, pp. 7263-7268. ).

Angiogenin is structurally homologous to the enzyme pancreatic RNase; Bovine pancreatic RNase A (RNNase), the most widely investigated protein, has a 34% have the same sequence (Strydom et al., 1985, Bi-ochewi stry, Vol. 24, pp. 5486-5494; Ku-rachi et al., 19 1985, Biochemistry 1, Vol. 24, pp. 5494-5499). a The tertiary structure of angiogenin consists of (i) three conservation of four disulfide bonds, (■ ) significantly dense binding to placental ribonuclease inhibitor (PH1) (5hapi ro and Vallee, 1987, Proc.

Natl, Acad, Sci, USA, Volume 84, Pages 2238-2241. ; Lee et al., 1989b 1Biochemistry, Volume 28, No. 2 pp. 25-230; Blackburn et al., 1977, J. Boil. Chew, Vol. 252, pp. 5904-5910) and (ffl) Computer - Fabrication of three-dimensional structures (Palmer et al., 1986, Proc. Natl. A. Based on CAD, Sci, USA, Vol. 83, pp. 1965-1969) Similar to the tertiary structure of RNNase. Three major catalytic residues of RNNase (■is-12, Lys-41 and +l1sl19) are many other key active parts. As well as positions and structural residues, angiogenin (■is-13, Lys-40 and Hisl14). Indeed, as mentioned above, angiogenin is Although it has polynucleolytic acid degrading activity, it is a completely different type of RNase from other RNases. Ru. The activity of angiogenin is greater than that of RNase against the most commonly used substrates. About 6 times lower (5hapfro et al., 1987, P-roc, Natl, A. cad, Sci, USA, Volume 84, Pages 8783-8787; 5hap fro et al., 1988, ^nal, Biochem, Vol. 175, No. 450 ~461 pages; Harper and Vallee, 1989, Biochem istry, Vol. 28, pp. 1875-1884). In this way, structural similarity also Regardless of the RNase (this RNase is also involved in angiogenesis and secondary messenger activity) angiogenin activity in vivo and in vitro (compared to The strikingly large differences clearly reveal distinctive physiological functions for angiogenin. clearly shown.

Recent studies have shown that the active site histidine and lysine residues of angiogenin are It was found that it is necessary for both tube formation inducing activity and liponucleoside decomposition activity (5h a-piro et al., 1986, Biochemistry, Vol. 25, No. 35 pp. 27-3532; 5 Hapfro et al., 1987b.

Proc, Natl, Acad, Sci, USA, Volume 84, No. 8783 ~8787 pages: 5hapfro et al., 1988b 5BiochetBio phys, Res, Commus, p. 156, p. 530-536; 5Hapfro et al., 1989, Biochemistry, Volume 28, No. 1 pp. 726-32; and 5hapfro and Vallee, 1989, Bio Chemistry, Vol. 28, pp. 7401-8). However, bovine pancreas R Nase A contains corresponding histidine and lysine residues but is angiogenic. Since there is no such thing, additional molecular features must be important for angiogenesis.

Such additional molecular features, if identified, would suggest that angiogenin It must contain regions of unique residues and/or sequences that are specific for. Spring Harper and Vallee (1989, Bioc hem, Vol. 28, pp. 1875-84) between angiogenin and RNase. One of the structural differences is the Cys-65-Cys-72 disulfide bond. We observed a virtual absence of sequence similarity within the regions of RNase containing RNases.

Angiogenin does not have this disulfide bond. This of angiogenin To identify the regions, Harper and Vallee ) (1989, supra) is a novel covalently bonded angiogenin/ It describes ARH-1, an RNase hybrid protein, and this In RH-I, a segment of angiogenin consisting of amino acid residues 58 to 70 is replaced by the corresponding segment (residues 59-73) of RNNase. Ta. This segment of angiogenin was compared with the corresponding segment from RNNase. By genetically replacing it with RNNase, it has properties more similar to RNNase. A covalently bonded hybrid protein was generated. ARH-I has significantly increased RNA ze-like enzyme activity, but clearly decreased angiogenesis-inducing activity. halper and Vourley (1989, supra) have increased angiogenic biological activity. It has not been possible to produce or identify a hybrid protein that does this. child Hybrids with angiogenic biological activities such as angiogenin This was unexpected given their experimental design, which incorporated an RNase-specific region. Taro.

Summary of the invention of residues 8-22 of angiogenin or corresponding to residues 8-22 of angiogenin Angiogenin-specific segment of angiogenin consisting of the N-terminal amino acids by replacing it with the corresponding segment (residues 7-21) of RNase. results in a novel recombinant hybrid protein with increased angiogenesis-inducing ability. This has now been surprisingly discovered.

According to the present invention, the N-terminal region of angiogenin is characterized by the unique potential of angiogenin. Regarding sites identified as candidate regions and thus specific for angiogenin associated as important. This area is divided into (1) four species (human, cow, pig, and It is highly conserved in the amino acid sequence of angiogenin (rabbit). and (2) different from the corresponding region of RNase. Novel recombination of the present invention ARH-1, a covalent angiogenin/RNase hybrid Thus, a segment near the N-terminus of angiogenin (residues 8-22) becomes RNN. by local mutagenesis with the corresponding segment of the enzyme (residues 7-21). It consists of the modified angiogenin obtained. This new recombinant hybrid is angioge 10 times greater angiogenic activity and placental ribonuclease inhibition compared to Nin showed increased binding (of the order of magnitude of at least 1) to substance 11 (PRI) . The liponucleolytic activity of ARH-1 did not change against many substrates.

However, the ability of ARH-m to suppress cell-free protein synthesis is limited by angiogens. This was a 20 to 30 times decrease compared to the standard.

Thus, a new hybrid containing 15 peptide segments of RNNase was created. biologically more active than angiogenin itself, and in fact enhances angiogenin. It's Genin.

Drawing description Figures 1a and 1b show angiogenin-specific segments as described in Example 1. To determine bovine angiogenin (Figure 1a) and RNNase (Figure 1b) A graph showing the percentage of amino acid sequence identity in the residue numbers of human angiogenin compared to This is a rough diagram. Figure 1c shows the difference in percent agreement between Figures 1a and 1b. It is a graph diagram.

Figure 2a shows the N-terminal amino acid of RNNase angiogenin and ARH-III. The amino acid sequence is shown. RNases retained in pancreatic RNases from 39 mammalian species Residues in ZeA are marked with an asterisk. The residues in angiogenin surrounded by a square are human, Retained in bovine, porcine and rabbit proteins. Figure 2b was described in Example 2. used as fragment B in the structure of the sequence encoding sea urchin ARH-III. Residues 4-23 of ARH-III encode a synthetic double-stranded oligonucleotide The DNA sequence of

FIG. 3 is a diagram of the structure of the sequence encoding ARH-I[[ described in Example 2.

Figure 4 shows the HP of tryptic peptides of ARH-III as described in Example 4. LC elution pattern is shown.

Figure 5a shows the activity of angiogenin and ARH-III on yeast tRNA. Figure 5b is a graphical diagram showing angiogenin and RN as described in Example 5. Figure 3 shows the effect of pH on the activity of ARH-n compared to ARHase.

Figure 6 shows the effect of angiogenin and ARH-m on intact liposomes. - Autoradiograph of a polyacrylamide gel (described in Example 6).

Figure 7 shows the activity of ARH-Iff and angiogenin in the CAM assay. (See Example 7).

Figure 8 shows protein iff: angiogenin and PRI conjugate over 10 days. FIG. 3 is a graph diagram showing the dissociation of ARH-ff.

FIG. 9 is a ribbon diagram of RNNase.

Detailed description of the invention Before describing the present invention, certain terms used herein will be defined.

Biologically active means that it is in a biological context (i.e., in an organism or replicated in vitro). A function or group of functions performed by a molecule. Regarding angiogenin biological activity is characterized by its angiogenic activity (angiogenesis) . Also includes secondary metsenger and/or ribonucleolytic activity.

Angiogenic activity is the chemical stimulation of blood vessel growth in tissues. This angiogenesis induction Activity was determined using the chicken fetal chorioallantoic membrane assay (Knighton et al., 1977, Br. , J, Cancer.

35, pp. 347-356) and/or rabbit corneal transplant assay (Lang et al. er and Folkman, 1976, Nature, vol. 263, no. 797 800 pages)).

Ribonucleolytic activity is the enzymatic degradation of RNA, and is limited to rRNA and tRNA. It also includes catalytic cracking.

Mutated genes or DNA structures have been modified by human intervention and are naturally occurring in ponds. DNA molecules or molecules that contain segments of DNA that have been transformed, joined, or juxtaposed in a manner that does not is a clone of such a molecule.

Mutated (mutated) angiogenin proteins are those in which one or more amino acids are compared to non-mutated or wild-type angiogenin angiogenin protein or any part of the protein that has altered biological activity when It is a peptide fragment of the meaning.

A preferred prokaryotic host cell for use in practicing the invention is the bacterium Escherichia coli ( Escherichia coli), and Bacillus and and other true strains.

These host cells are transformed and the cloned foreign gene is expressed within them. Techniques for this are well known in the literature (e.g. Ilaniatis et al., 1999). 1982, Ma-Iecular Clonjng; A Laboratory Manual, Co1d Sp-rfnger Harbor Labor atory, Co1d Spring Harbor, New York:C current Protocols in 1 lolecular Bj, -o logy, 1987 (latest information until 1989), Au5ube1, etc., G reen Publishing As5ociates and Wiley -Interscience, New York; Perbal, 19 1988, Ap-ractical Guide to) lolecular Cloning, John filey & 5ons, New York ; Davis et al., 1986, Ba5ic l1e-thods in M o1ecular Biology, Elsevier SciencePub lishing Co., New York) a foreign genes in bacterial host cells. Vectors used to express genes are generally labeled with a selectable label, e.g. Contains genes related to biochemical resistance and promoters that function in host cells . Preferred promoters are trp (N1chols and Yan~ofsky, 1983, Ieth, in Enzya++ology, Volume 101, No. 1 55 pages), lac (Casadaban et al. 1980, J. Bact , Vol. 143, pp. 971-980) and the phage human promoter system. Contains. A preferred plasmid for transforming bacteria is pBR322 (Boli var et al., 1977, Gene, vol. 2, pp. 95-113), pUC pla Sumido (by Messing, 1983, Meth, in Enzymo1o gy1 volume 101, pages 20-77: and by Vieria and Messing, 1982, Gene, Vol. 19, pp. 259-268), pCQv2 (Qui John, 1983, J, 1lo1. Appl, Genet, Volume 2, No. 1 to 10) and derivatives thereof.

Eukaryotic microorganisms, such as yeast, Saccharomyces cerevisiae (Saccharo@y ces Cerevicise) can be used as host cells. yeast Procedures for transforming are also known (eg, 5trathern et al. Author, 1982, The 1 locular Biology of th e Yeast 5a-ccharoBces, Volume 2, Cold Sprin g Harbor Laboratory, New York; Per bal, 1988, supra; Dav-is et al., 1986, supra; Be ggs, 1978, Nature, Vol. 275, pp. 104-108) . Expression vectors for use in yeast are YEI) 13 (Broach et al., 1979, Gene, Vol. 8, pp. 121-133), Y Rp 7 (Struh1 et al., 1979, Proc, Natl, cad, Sc i, USA, Vol. 76, pp. 1035-1039), pJDB248 and p. JDB 219 (by Beggs, supra) and its derivatives. Let's sled Such vectors generally carry a selectable marker, e.g. the trpl mutant gene. Contains a nutrient-tagged TRP that allows selection in different host strains. yeast expression vector An advantageous promoter for use in the yeast glycolytic gene et al., 1980, J. Biol, Chet, Volume 255, No. 12073 ~12080 pages; ^1ber and Kawasaki, 1982, J, 1lo1. A ppl, Genet, Vol. 1, pp. 419-434) or alcohol dehydrogenation. Genes (Young et al., Genetic Engineering of i licroorganisms for Ch-emicals, Holla ender et al., p. 335, Plenum, New York, 1982; and Ammerer, 1leth, in Enz-y■oiogy, No. 10 1, pp. 192-201, 1983). yeast Facilitates the purification of angiogenin protein produced in the transformants and A yeast gene encoding a protein that is advantageously secreted to obtain sulfide formation. from the offspring, the signal sequence binds to the sequence encoding for the angiogenin protein You may do so. A particularly advantageous signal sequence is the pre-pro region of the MFα1 gene ( Kurjan and Herskovitz, 1982, Ce1l, Volume 30 , pp. 933-943).

Advanced eukaryotic cells can also be used as host cells in practicing the invention. Cultivation Cultured mammalian cells are advantageous. Expression vectors for use in mammalian cells are Promoter capable of directing transcription of foreign genes introduced into mammalian cells Consists of - A particularly advantageous promoter is mouse metallothionein-1 (MT- 1) It is a promoter (Pal++1ter et al., 1983, 5cien ce, Vol. 222, pp. 809-814). Similarly, contained in the expression vector A polyadenylation signal is located downstream of the insertion site. polyadeni The angiogenesis signal may be a signal of a cloned angiogenesis-inducing protein gene. or may be derived from a heterologous gene. Other genomic sequences that enhance expression (e.g. For example, intronic sequences) may be inserted.

The cloned gene sequence is prepared by methods known in the art, such as calcium phosphate-mediated transcription. introduced into cultured mammalian cells (Wigler et al., 1978, Ce1l , Volume 14, Page 725; Coraro and Pearson, 1981. So-matic Ce1l Genetics, Volume 7, Page 603; Gr aha+ and Wander Eb, 1973, Virology, No. 5 Volume 2, page 456). The precipitate is formed from DNA and calcium phosphate, Apply this precipitate to the cells. Some cells take up DNA and carry it Keep it inside the cells for several days. A small amount (typically 1O-4) of these cells integrates DNA into the genome. To identify these integrants, selectable A specific phenotype (selectable marker) is generally introduced into the cell together with the gene of interest. It will be done. Advantageous selectable labels include drugs such as neomycin, hygromine and and genes that confer resistance to methotrexate. Selectable signs may be co-introduced with the gene of interest on an isolated plasmid into cells. or may be introduced on the same plasmid.

The number of copies of the integrated gene sequence is determined by a fixed selectable marker (e.g. methotrexate). A conventionally known method using dihydrofolate reductase (which confers resistance to xate) It can be increased by amplifying with .

This selectable marker is introduced into the cell along with the gene of interest, and drug selection is made as appropriate. used. This drug concentration is then increased in a stepwise manner, selecting resistant cells at each step. I will. In selection for increased copy number of cloned sequences, expression levels may have increased sufficiently.

The mutant angiogenin protein produced according to the present invention can be used in clinical pharmaceutical preparations. diagnostic products or by combining with suitable carriers. It can be used for various purposes. Contains this protein in an amount effective for inducing angiogenesis. These pharmaceutical compositions are also encompassed by the present invention. Such pharmaceutical compositions may be administered to mammals, For example, to promote or influence the growth of the vascular system in cats, dogs and humans. for example, to induce collateral circulation after a heart attack or other ischemic heart disease. can be used. For this purpose, the angiogenin protein of the present invention is Administered orally. As used herein, the term “parenteral” refers to subcutaneous, intravenous, intramuscular Includes intramuscular or intrafibular injection or infusion methods. Applicable acceptable vehicles and solutions Examples include "water for injection," Ringer's solution, and isotonic sodium chloride solution. Ru. In a typical implementation, the selected protein is added to the vehicle, typically water for injection. The protein containing an effective amount of protein for inducing angiogenesis is dissolved in Preparation for parenteral administration in dosage units containing: The effective dose is determined to achieve the desired clinical effect. It is necessary to adjust to each individual situation by clinical admission until it is decided.

In order to widen their therapeutic spectrum, these pharmaceutical compositions can be combined with other drugs, e.g. - adreceptor antagonists, e.g. propranolol, calcium channel blockers; Withdrawals, such as nifedipine or other coronary artery dilators They can also be administered simultaneously.

The proteins of the invention can be used to treat wounds, e.g. in joints or other locations involving soft tissue. It can also be used to promote. For example, if the injury is a sports-related injury or if it occurs in the meniscus of the knee or shoulder, as often occurs in osteoarthritis, there is no cure. Long-term release implantation of a therapeutically effective amount of angiogenin protein at the site of injury is an advantageous procedure for the treatment of traumatized fibrocartilage material. Protein of this invention The substance can also be administered topically to the wound site. For topical application, the present invention Selected angiogenic proteins are combined with dermatologically tolerable substances such as petrolatum and talc. Mix with carrier and apply liberally to area of action. using recombinant DNA technology Local mutagenesis is performed to produce the mutated protein according to the present invention in the amount necessary for clinical application. provide an excellent way to produce. Angiogenin and RNNase It is sufficient to show similarities in amino acid sequence, three-dimensional structure, and RNA cleavage ability. It is publicly known. Despite this, RNNase is not angiogenic (endothelial did not induce a secondary mesengen response in cells (Bic-knell and Vall ee, 1988, Proc, Natl, ^cad.

Sci, USA, Vol. 85, pp. 5961-5965) and complete liposomes. (St. Clair et al., 198 7th year, Proc, Natl.

^cad, Sci, USA, Volume 84, Pages 8330-8334: St. Claire et al., 1988, Biochemistry, Vol. 27, No. 726 3-7268). In vivo and in vitro activity of angiogenin on RNNase Because the activities are clearly different, the molecular features that form the basis of these activities are also different. It should be. In order to identify such characteristics, samples from different species are Naturally occurring sequence variations in giogenin were analyzed as described in Example 1. the Regions of angiogenin that are important for specific activities are (i) conserved across species; (if) should be different from the corresponding region of the RNase. Ru. According to Example 1, if such regions are identified, the factual soil of these regions The role of cleavage can be investigated using local mutagenesis as described in Example 2.

Sequence of human angiogenin and bovine angiogenin (Figure 1a) and bovine pancreatic 11R Comparison with the sequence of Nase A (Figure 1b) produced a similar plot.

The difference between the two plots, 1a minus lb, indicates that this region of human angiogenin is Having both high identity to angiogenin and low identity to RNNase. (Figure 1C). From this analysis, one segment stands out. Extracted: This was a segment centered on residues 16-19. This analysis applied a 5-residue “window”, so the actual amino acids included are Tyr-14 or It is ＾rg　-21. These residues (other than Tyr-14) in the three-dimensional structure ) is an extension extending straight from the active site based on the comparison with RNNase (Figure 9). Located in the long structure. Further investigation of this amino acid sequence (Fig. 2a) Six of the eight residues in mammalian angiogenin have so far been sequenced. It shows that it is kept inside. Only one of these eight residues, ^ sp-15 is also present in RNase A. Thus, this sequence This corresponding sequence in pancreatic RNase is mostly It is not conserved across species (Fig. 2a). In fact, Hofmann et al. -ann et al., 1966, J. Amer, Cher, Sac, Vol. 88, (pages 3633-3639) that residues 15-20 are not essential for RNNase activity. It shows that there is no. Conserved in four species of mammalian angiogenin The block of amino acids from His-8 to His-13 is the remainder of angiogenin. It adjoins and precedes groups 14-21 (Fig. 2a). Residues 8-13 begin with 5er-4 It forms an α-helical part and assumes a similar structure on RNNase (Figure 9). 2 Residues His8 and Thrl1 are retained in 39 mammalian pancreatic RNases. and replace the RNNase-resistant Glu-9 with a hydrophobic block. Change depending on Ishin. Therefore, residues Hls-8 to His-13 are RNN ase showed potentially significant differences. Therefore, this complete reserve of angiogenin To elucidate the functional role of residues 8 to 21, we combined them with residue 22 (protein studied using local mutagenesis (to preserve the original net charge of ).

Local mutagenesis differs from site-specific mutagenesis in that it involves mutagenesis of a single residue rather than a single residue. Rather, segments or regions of amino acids are replaced. This means that there are many residuals at the same time. It allows the testing of groups and also the study of their contribution to secondary structure. a Due to the tertiary structural similarities between angiogenin and RNNase, the selected regions Even if a region is replaced from one protein to another, the overall structure is retained. There is. The effectiveness of this research method is that residues 58-70 of angiogenin are isolated by RNase 5. 9 to 73 (Har-per and Valle e, 1989, Biochemistry 1 Vol. 28, No. 1875-188 4 pages). The resulting mutant hybrid protein was named ARH-I and was The three disulfide bonds found in giogenin as well as the RNNase The fourth disulfide bond from was formed similarly and folded into the correct structure. . Thus, in particular, a peptide consisting of amino acid residues 1 to 21 of angiogenin^n g(1-21) is a peptide RN consisting of amino acid residues 21-124 of RNase A micromolar dissociation constant is non-covalently bound to the enzyme (21-124); 50% of the enzymatic activity of RNNase was reconstituted during (Flarper et al., (1988, Bioch-esistry, Vol. 27, pp. 219-226) Considering that the N-terminal region of angiogenin also shows the overall structure It would be possible to retain and easily replace. This is true throughout this area. showed that there is structural similarity between the two protein questions. Amino terminal of RNNase The end regions have undergone extensive crystallography and chemistries due to the assimilation of the S-peptide/S-protein system. It was the subject of academic research. The term S-peptide refers to a limited subset of RNNases. RNase peptide consisting of residues 1 to 20 produced by tilisin proteolysis. related.

The remainder of the molecule, consisting of residues 21-124, is called the S-protein (Richard s and Vithayathill 9, 59, J.

Bfo, Chet, Vol. 234, pp. 162-166). Figure 9 shows the RNN a A ribbon diagram of the enzyme is shown, showing the positions of essential active sites, His-12, Lys-41 and and His-119 are prominent. Similarly, look at Lys 7 and 5et-21. It's coming out. As shown in Figure 9, residues 3-13 of RNNase are active site cleavage. 1is-12 and substrate-binding Gin-11 residues, which are in contact with the forms an α-helix; this helix has submicromolar resolution in the S-protein portion of the molecule. non-covalently bound with a discrete constant (Richards and Wyckoff, 1971; Enzymes (3rd revised edition), Volume 4, pp. 647-806 and references therein. References). Residues 15-23 extend away from the active site and around the outside of the molecule, and Form a junction with the second α-helix. Many residues are endowed with specific functions. (Richards and Vyckoff, 1971, supra; Rich ards and fyckoff, 1973, At1as of Mo1ecu lar 5 structures in Bi.

1agy (Phillips, D, C, and RichardslF, I , Publishing), Volume 1, Oxford University PressSLon Don; Blackburn and Moorell 982, Enzy■e s (3rd revised edition), Volume 15, pp. 317-433; written by Flodaver et al. 1982, J. Biol, Chew, Vol. 257, No. 1325-13. 35 pages), Glu2 (side chain hydrogen bond (H-bond) to ^rg-10, Phe -8 (hydrophobic interaction with protein core), Arg10 (side to Glu-2) chain H-bond), Gln-11 (side chain H-bond to substrate phosphate: A>n -4 backbone carbonyl H-bond to 4), His-12 (involved in catalysis) side chain; H-bond to carbonyl of Thr-45), Met-13 (protein covalent hydrophobic interaction with A) and ^sp-14 (Tyr-25 and/or Arg side chain H-bond to -33). The importance of these residues is given by let-1 All but 3 are retained in pancreatic RNases of 39 different mammalian species. This is reflected in the fact that Bi.

1, Evol, Volume 3, pp. 262-275). On the contrary, residue 15 ~23 are not conserved among these different RNases, and none of the aforementioned As such, residues 15-20 are completely non-essential.

The amino-terminal region of angiogenin has, by comparison, been less intensely studied. do not have. However, recently the amino acid sequence analysis of angiogenin from several species has been carried out. achieved. The sequence of the N-terminal region of angiogenin is shown in Figure 2a: four variants. Residues that are retained in different species are boxed. Angiogenin from these various species Sequence analysis of angiogenin shows that 16 of the first 21 residues of angiogenin are unchanged. 12 of the 16 residues differ from their corresponding residues in RNase A. It has been made clear that It is retained in all pancreatic 11 RNases. Mino acids are replaced in angiogenin: these are Glu-2,^1a -5, Lys-7 and Arg-10. RN Ah+! Comparison with the structure of A In , residues 4-14 of angiogenin are oriented in the active site section with Glu- will form an α-helix with side chains of 12 and 1lis-13. next , residues 15-24 are arranged in several strands of the β-sheet in a relatively unordered manner. It extends toward the “back side” of the molecule, at the rear of the molecule, and bonds with the second α-helix. Book The region selected for mutation according to the invention consists of residues 8-22 of angiogenin. and corresponds to the sequence of RNNase ranging from residues Lys-7 to 5et-21. , clearly shown in FIG. Genetically engineered hybrid called ARH-III The structure of the DNA encoding sequence for the protein is described in Example 2. A.R. The coding sequence for H-H was expressed in E. coli host cells and ARH-m The protein was purified as described in Example 3. AR) Code regarding I-H sequences in cells other than bacteria, such as yeast or mammalian cells as described. It can also be expressed in host cells. ARH- purified from E. coli expression system 1 [1 Protein, amino acid analysis and trypsin peptide analysis (Example 4) Residues 8-22 of genin were successfully replaced with residues 7-21 of RNNase. , further consisting of the removal of 1let-(-1) from the E. coli expressed protein.

Local mutagenesis can be used to generate hybrids for most RNA substrates as described in Example 5. It has almost no effect on the activity of the protein ARH-III. At pH 6,8, A RH-■ cleaves tRNA at the same rate as angiogenin (Figure 5a), However, there was a slight deviation at the highest pH value (Figure 5b). Ginucle Otide RNA substrate (Sigma Chemical Co or Calbioch em-Beh-ring), cytidylyl-(3'-5')a denosine (CpA, ), ditidylyl (3'-5') guanosine (CpG) , uridylyl-(3'-5') adenosine (UpA), and uridylyl-( 3'-5') 18S and 2 isolated in the same manner as guanosine (UpG) (Table ■) The cleavage rate for 8S rRNA is also essentially the same as that for angiogenin. It was. Therefore, this replaced region is responsible for this activity in angiogenin. It was thought that they did not play an important role. Furthermore, these results suggest that angiogenin The relative difference in dinucleotide preponderance that exists between RNNase and RNNase is ( 5hapiro et al., 1988, Anal, Bi.

c-hem, Volume 175, Pages 450-461, Harper & Va-11 ee, 1989, Biochemistry, Volume 28, Nos. 1875-188 (page 4) It is said that it is probably not bound from substrate-enzyme interactions with any of these residues. suggests that. The lack of effect on general RNase activity may be due to angiogenic does not interfere with its role in liponucleic acid degradation in its mechanism of action; This suggests that the degradation involves specific substrates.

In contrast, as described in Example 6, local mutations inhibit protein synthesis and ARH-■ in induction of formation and also in secondary Messenger assays It had a clear effect on the activity of ARH-II inhibits cell-free protein synthesis. It had about 30% more activity than angiogenin; this effect was due to the increase in total protein content. Incorporation of L53-methionine (Table 1) and specific rR from intact liposomes Both production of NA cleavage products (Figure 6) was observed. It is stated in example 8. In the CAM angiogenesis assay, ARH-111 showed a factor of 10. It had unexpectedly greater potency than angiogenin (Figure 7). ARH-11 1 reaches sufficient activity at 0.1 ng per egg, and at this time angiogenin is semi-required lng (Fig. 7). Similar results were obtained with secondary metusene in endothelial cells. It was also observed in the jar assay. Release of diacylglycerol or prosthesis The dose-response curve for ARH-1, which induces the secretion of tacycline, is The concentration was 10 to 100 times lower than that of ogenin.

Effect of local mutations on angiogenesis (10-fold increase in potency) and protein synthesis Correlation between the effect of local mutations on inhibition (30-fold reduction in potency) The lack of suggests that these two activities are not coupled. These 2 The two activities represent separate and unrelated functions of the angiogenin molecule. Opposition , the correlation between the effects of local mutations on angiogenesis and secondary metsenger activity. The relationship suggests that these two activities of angiogenin may be coupled. ing. Regarding these two activities (angiogenesis and secondary metsenger), ARH The dose-response curve for -H is 10-100 times lower in concentration compared to angiogenin. Move every time. These data suggest that the coupling between these two activities is Supports the hypothesis that this may involve activation of genin receptors.

As described in Example 8, ARH-m has a 1. shows increased binding at levels of . PRI is a close link between angiogenin and RNase. It is a binding inhibitor. This 50kDa protein is angiogenic with a Ki of 0°7fM. (Lee et al., 1989b, Biochemistry, Volume 28, pp. 22'5-230). The interaction minimally surrounds the active site and T disturb the region around rp-89 (Lee et al., 1989a, Bi-och e-kai 1stry, volume 28, pages 219-224 + Lee & Va-11ee , l9g9a, Biochemistry, Volume 28, No. 355 6-3561). PRI and RNN interact 60 times weaker than with angiogenin interaction with Aase (Lee et al., 1989b 5Bioche shadow, Bio phys, Res, Co■■un.

, Vol. 161, pp. 121-126) without significant contribution from residues 1-20. It has been reported that it occurs only within the S-protein portion of the molecule (Blackbu rn & Jai-1khani, 1979, J. Biol, Chewl. , Vol. 254, pp. 12488-12493). Therefore, the angiogear according to the present invention Local mutation of Nin residues 8 to 22 indeed results in PRI as shown in Table V. It is surprising that it increases the affinity of ARH-111. The meeting speed is The dissociation rate decreased by a factor of 4 to the limit of detection, i.e. to the limit of detection. . Therefore, Ki is at least 1 greater than the Ki for angiogenin. The extent of this is low. This change is due to the enhanced angiogenesis-inducing activity of ARH-III at low concentrations. This was unexpected in terms of both sex and cell response activity.

In the present invention, in addition to research involving mutagenesis, suitable reproduction of mutant proteins will be carried out. Demands regarding life are increasing. Errors regarding ARH-III local mutants There are three pieces of evidence regarding the form that was passed on without exception: (1) All three jisuru The fido bonds are precisely formed, and are widely distributed (from isolated positions) in the sequence. requires the exact sequence of six cysteine residues: (2) four dinucleos; The rate of hydrolysis of Tide and other RNA substrates is unaffected by local mutations. This is due to mispositioning of different residues that are coupled to the phosphodiester bond cleavage. and (3) ARH-III is not significantly communicated to the PRI. Tightly bound: this interaction still requires precise three-dimensional arrangement of multiple residues and this residue is included in the conjugate and has a low Ki value (≦7×10-17M ) is not consistent with large selectivity of shape.

The results were compared to the novel angiogenin/RNase hybrid protein ARH- The characteristic angiogenin activity of 1t [ The following examples demonstrate that . In this way, the present invention will be explained in more detail by examples. The invention is not limited.

Example 1 Selection of regions for mutagenesis Sequences of human angiogenin and bovine angiogenin (Fig. 1a) and RNN Analogous protocols were used to provide a quantitative means of comparing the sequence (Figure 2b) to The total amount was calculated. This plot is centered on the angiogenin residue that is pointed out. Percent identity between two proteins within the combined moving 5-residue window is shown. this is doctor - D. J. Strydam; Harvard Medical It was carried out using the computer program services of the School of Engineering. array To facilitate alignment of the RNase residues, ^sn-24, ^rg-39, G in-69, Thr-70, Pro-114 and TYr-115 (RNN Deleting the N-terminal alanine of bovine angiogenin as shown in Figure 7) I let it happen. This value is averaged over five residues, so this plot Emphasizes local trends rather than similarities/differences at different locations.

This first plot compares the sequence of human angiogenin and bovine angiogenin. (Figure 1a). In many cases, the identity is greater than 60%, with a total amount of 64% - (Maes et al., 1988, FEBS Lett.

, Vol. 241, pp. 41-45, Bond and Strydom, 1989, B iochemistry, Vol. 28, pp. 6110-6113). amazing thing However, the three essential catalytic residues of angiogenin, His-13, Ly The region around s-40 and ■1s-114 has high similarity to protein questions: In fact, 7th to 19th, 37th to 48th, and 106th to 114th positions have more than 80% identity. The three longest segments were generated. 5 different regions are 100% identical The longest segment was the 10th to 16th segment. Figure 1b is human Angi. A comparison is made between ogenin and RNNase. The degree of identity is what we observed earlier. The total sequence identity is generally low, consisting of 33%. In this case, what you are most interested in is actually This is the difference between these two sequences. This is because this difference is due to the different proteins. This is because it seems to play a biological role. The least similar short? The regions (0% identity) are 4th to 6th, 50th to 52nd, 64th to 65th, and 100th to 65th. Occurring at position 102; the longest region was 18-22.

Figure 1c shows the difference between the two figures, from the corresponding values in Figure 1a to 1b. It was calculated by subtracting the value from the figure.

The high values in Figure 10 indicate high homology with bovine angiogenin and a high value for RNNase. Regions of human angiogenin with low identity are shown. These are Angioge This is the area most likely to be related to the unique role of Nin. four separate windows The injuries are distinctive in 80% identity, and these three are 16.18 and 1 At 9, it occurs nearby. Thus, this one small segment It is most conspicuously retained in RNNase, and most different from RNNase. is. The residues contained within this segment include all of the two terminal windows. Tyr-14 to ^rg-21. − Figure 2a shows the N-terminal sequence of human angiogenin for RNNase. Mammal 39 Residues in RNase A that are retained in pancreatic RNase from species (Be1nte■ a et al., 1986, l1o1. Riot, Evo, Volume 3, No. 262~ (page 275) are indicated by an asterisk. The boxed residues in angiogenin are Humans, Cattle (Bond and Strydom, 1989, Biochemistry , Vol. 28, pp. 6110-6113, Maes et al., 1988, FEEB S Lett.

, Vol. 241, pp. 41-45) and is retained in pig and rabbit proteins. Q is It is pyroglutamic acid.

In particular, Figure 2a shows that immediately before residues 14-21 of angiogenin are mammalian angiogens. A block of amino acids 1lis-8 to His-13 retained in 4 types of amino acids. This residue forms the part of the α-helix starting at Ser-4. The structure is assumed to be similar to RNNase as shown in FIG. two residues , His-8 and Thr-11 are retained in 39 mammalian pancreatic RNases. The acidic Glu-9 is replaced with the hydrophobic leucine to the RNNase. replace. Therefore, these residues also showed clear differences in RNNase. subordinate So, to explain the functional role of this complete section of angiogenin, Residues 8-21 were removed by local mutagenesis (retaining the original effective charge of the protein). ) was investigated together with residue 22.

Example 2 Structure of the sequence encoding ARH-m In example 1, the complete section of angiogenin extends from His-8 to ^5p22. selected for mutagenesis by Residues 7-21 to RNNase (Figure 2a). Hive called ARH-m (Fig. 2a, b) The sequence encoding the DNA for lid protein is shown in Figure 3 as follows. It was configured as follows. Fragments A and C are pAng2 DNA (13μIF ) is cleaved with Who I and EcoRI or Pvu I and EcoRI, and then NuSieve G T G low melting point agarose (FMCBioproduct prepared by electrophoretic purification in a 3% gel using did. DNA from expression plasmid pAng2 was obtained from Shapiro. (1988, ^nal.

Biochem, Vol. 175, pp. 450-461 and U.S. Patent No. 30596. 8, which is a reference). Plasmid A-g2 is a modified Tr Includes p-promoter and ampicillinase tag for selection.

Fragment A (2928 bp) contains the complete non-coding region of the plasmid and l1 Fragment C includes the coding region from et-(-1) to ^sn-3: ^rg-24 to Pro-123 coding region and includes two stop codons. Ru. Fragments A and C are then combined with the residues of angiogenin as shown in Figure 2b. 4-7, a synthetic protein encoding 7-21 of RNase A and 23 of angiogenin. It binds to fragment B, which is a full-stranded oligonucleotide. This synthetic oligonucleotide The end of leotide is compatible with the ligation of Who I and Pvu I restriction enzyme cleavage sites. There is. This synthetic double-stranded oligonucleotide was produced by Biopolymer Laboratory (Bi opolymer Laboratory SDept of Biolo-gi caL Chemistry and Mo1ecular Phar+*ac ology.

11arvard Medical 5chool). Each chain is β- Milligen Synthesizer using cyanoethyl phosphoramidite automatic synthesis method (IlilLigen 5ynthesizer) according to conventional methods. (by Atteuccf and Caruthers, 1981, J. ser, Chew, Soc, Volume 103, Pages 3185-3191: l]e Hucage and Caruthers, 1981, Tet, Letter s1 Volume 22, I! 1859-1862). Duplex is Halper (t larper and Vallee obtained by annealing 5′ phosphorylated complementary oligomers ( (1989, Biochemistry, Vol. 28, pp. 1875-1884) . The most successful coupling reactions involved fragments A, B and C24,150 and Apply 100 μL and 6 units of T4 DNA ligase in a 60 μl volume; final Agarose concentration was 1.4%. E. coli strain W3110 cells were then incubated with Ca( , /2 method for transformation using the 1la-niatis et al., 1982, 1lo regular Cloning: ^ Lab-oratory 1lanu al, Col 1d Spring Harbor Laboratory SCol d Spring Harbor, New York) and plasmid incorporation This was detected by ampicillin resistance.

Restriction enzyme mapping (using Haem or EcoRI and KpnI) and Nucleotide sequencing and analysis are both methods for new genetically engineered plasmids, the so-called p applied to ensure that ARH-III contains the correct coding sequence. . DNA sequencing was performed using [3158]-deoxyadenosine-5'-(α-1000) Sequenase 1 kit (U, S, Biochemi) with rephosphate calCorp.

) was carried out. For sequencing, [[pn I - The EcoRI fragment was isolated and placed into Ml 3mp 18 using conventional methods. Clone. The nucleotide sequence corresponding to the first 60 amino acids of a protein is This was expected for a hybrid protein. Plasmid pARH-I II has been deposited with the United States Depositary (A, T, C, C,) under deposit number 68188. Expression and purification of angiogenin and ARH-m were performed by Shapiro et al. Described in detail (Shapiro et al., 1988, AIIal, B. iochevl, Vol. 175, pp. 450-461). easy Then, E. coli strain W3110 cells containing pAng2 were grown in supplemented M9 medium. , and then by addition of indole-3-acrylic acid. Next, the thin Cells were collected by centrifugation and suspended in lysozyme, NaCl, EDTA solution for 45 minutes. The mixture was turbid and then sonicated. Insoluble angiogenin is reconstituted by centrifugation. and recovered, dissolved in guanidine HCI and 2-mercaptoethanol, and then Regenerated by dilution into Tris-NaCl-buffer. Amicon (A■ 1con; Danvers, M.A. O1923) concentrated in a compression filtration apparatus. Then, sequentially applying angiogenin to the mono S and C18 reverse phase HPLC columns. It was purified by Furthermore, angiogenin was described in detail by Kurachi et al. Baby hamster kidney (BHK) cells genetically engineered as described in detail. (1988, Biochesi-stry, Vol. 27, No. 6557~ 62 pages). Briefly, transfected cells were incubated with 480 μM ZnSO. This was carried out in DME medium containing CdSO and 42 μM CdSO. Cultures were cultured at 37°C with exchange at intervals of 2 to 3 days. Then, angiogenin was converted to C Continuous chromatography was performed on M-cellulose ion exchanger and Mono S columns. It was purified from the medium by Used in subsequent steels unless otherwise noted Angiogenin was obtained from BHK cells as outlined.

B, ARH-m Separate cells of E. coli W3110 transformed with pARH-III as described in Example 2. The level of expression in nine colonies of 8, supra), immunoblotting using affinity-purified antiangiogenin. It was evaluated by ting. Cells showing the highest expression level (~10+g/j) were selected for ARH-■ expression.

Large-scale expression and purification was performed as previously described for angiogenin by Shapiro et al. (1988, supra). Thus, as described by Shapiro et al. Uni (1988, supra) hybrid ARH-III protein was injected into Escherichia coli W311. expressed in 0 and regenerated into monoS cation exchanger and Cl8 (octadecyl run). ) Purified using reverse phase HPLC. 5EP-PAK C18 cartridge is mm Millipore Carp.

, Bedford, MA O1730). ARH-11ha Hybrid proteins eluted earlier than angiogenin on the Mono S column (at retention time). 8 min difference between 0.15 and 0.55 M NaCJ slope in Tris lQmM , 50 minutes). A sharp contrast on the 018 column with a small shoulder that makes up 5% of the area. It eluted as a typical peak. Collect the central fraction of the larger peak, and Dialysis and 5Ds-PAGE analysis of single confluent cells running at Mr~14500 This shows the band. The final yield was 0.4+n per 11 cultures.

Net-(-1) residue (9 μM) of E. coli-expressed ARH-m hybrid protein As described by Shapiro et al. (1988, supra), the following residue Gln Aeromonas -1 under suitable conditions for automatic cyclization to pyroglutamic acid. (^ero+gonas) Enzymatically removed with aminopeptidase. Next, the egg The white matter was purified by reverse-phase HPLC as described above, the best fractions were collected, and water Dialyzed against.

Mutant or natural (non-mutated) expressed in E. 8I bacteria using pA1192 All angiogenins contain Met-(-1) at the amino terminus; Extra residues have not yet been found to affect enzymatic or angiogenic activity. Not yet. However, due to the proximity to the replaced region in ARH-m Then, the N-terminal Net-(-1) residue was removed as described above and the new region bound. The binding and orientation should be as close as possible to that of angiogenin. I made sure.

Amino acid composition and peptide mapping revealed that Met-(-1) in ARH-1 [[ 95% as described in Example 4 below.

Example 4 The structural characteristics of ARH-m are Amino acid analysis was performed as described (Strydos et al., 1985, Bi chemistry, Vol. 24, pp. 5486-5494) Picotag induction method ( Picotag derivati-zation method) and water association (Waters As5ociates; 11i1ford, MA 0175 7) It was carried out using the HPLC system. Tryptic digestion is performed using ARH- [14, in 0.35M NaCl, pH 8, constant temperature at 37℃ for 24 hours in 0730tll It was carried out by holding. Peptides according to Shapiro et al. (1988, supra) Therefore, the elution position and amino acid composition were separated by reverse phase HPLC as described. It was identified by Artex Ultrasphere (^1tex ultras) phere) IP C18 column was prepared using solvent A (0.1% trifluoroacetic acid aqueous solution). (TF A)) in solvent B (3:3:2.2-propertool:acetonitrile) :0.08% TFA) over a period of 140 minutes with a linear gradient solution consisting of 0-50% was used. The flow rate of the column was 0, 8817 minutes ◇5DS-PAGE Laesmli (1970, Nature, Vol. 227, No. 68) 0-685) with a 5% thickening gel. Performed on a lyacrylamide gel. Proteins are manufactured by ICN Immunobiological (Im Rapid Silver Staining Kit (Lislell L) from Munobiological , 60532).

The amino acid composition of ARH-1 is based on the residues of angiogenin as shown in Table 1. Successful replacement of RNase A at residues 7-21 of 8-22, as well as l1et It was consistent with the removal of -(-1). The results of tryptic digestion of ARH-I [[ are also shown in Table 1. and supported the successful substitution of residues 8-22 as shown in FIG. In particular, the Figure 4 shows the HPLC elution form of the tryptic peptide of ARH-■. individual pepti The DNA was identified by the amino acid composition revealed for the complete sequence. Angi Regarding Ogenin, the name of the peptide is Strydom et al. (198 5, Biochemistry, Volume 24, Pages 5486-5494) It is something. angiogenin and AR) (-■ N-terminal sequence is shown in Figure 4. : Double-sided arrows indicate peptides generated by tryptic cleavage. all three A pair of distin-binding peptides, T-9b, T-10 and T-11, were recovered and the protein Consistent with quality proper regeneration and oxidation. Peptides T-1, -2, -3a.

-3b, -4a, -5, -6, -8 and disphite bonds T-10 and T-11 are with the same composition and elution position as observed for human angiogenin (Str ydom et al., 1985, supra: 5hapiro et al., 1988, supra). a The mino-terminal peptide T-1 was recovered with a yield of over 95% and again Met-(- 1) showed essentially complete removal.

As expected, the difference between ARH-1 [[ and angiogenin 7 and T-9'. At that location, the ARH-1111 hybrid , two new tripeptides (termed T-7a and T-7b) as shown in Figure 4. ) and an extended form of T-9' (referred to as T-9b'). T-7a is the other 3 two or more poorly resolved 44 and 45 min along with one peptide. Peaks eluted; these were identified by amino acid composition. Peptide T- 7b (Table 1) has clearly distinguished peaks at 56 minutes as shown in Figure 4. It eluted as a block. The non-hydrolyzed hexapeptide that constitutes T-Tab is similarly processed. (Figure 4, Table 1). disulfide bonded to T-9' and called T-9b T-9b' forming a double peptide was detected in two forms. First, the conclusion Synthetic peptide T-9b eluted in essentially pure form with a peak at 111 minutes (Figure 4). , Table 1). Second, the large peak at 109 min is associated with T-8, T-9b and ^rg residues. Contains a mixture of groups 1-2. This is from the carboxyl end of T-9b' to T-1 3 (^rg-^rg peptide) and T-9b'-13/T-9'' Consistent with simultaneous elution of double peptide and T-g.

Table I Amino acid composition of ARH-III and its trypsin protein Angiogenin ^RH-m T-7b T-7ab T-9b^sp 15 14.0 (14) 0.14 2.19 (2) Glu 10 9.9 (10) 1.00 (1) 0.98 (1) 2.95 (3) Ser 9 12.6 (13 ) 0.12 6.56 (7) Gay 8 7.5 (7) 0.12 0.52 f(is　6　4.9(5)　1.11(1)＾rg　13　13.3(13) 1.00 (1) 1.03 (1) 2.17 (2) Thr 7 7.3 (7) 1.00 (1) 2.87 (3) Ala 5 6.1 (6) 1.87 (2) Pro 8 7.5 (7) 0.18 Tyr 4 2.7 (3) 0.95 (1) 0.86 (1) Val 5 4. 0 (5) 0.94 (1) Met 1 2.0 (2) 1.81 (2) 11e 7 6.5 (7) 1. (13 (1) Leu 6 5.3 (5) 0.1 0. 15Phe 5 4.9 (5) 0.90 (1) 0.90 (1) 0.98 ( 1) Lys 7 7.1 (7) 1.04 (1) 1.23 (1) Cys 6 6 (6) 2 (2) peel 52Q 45017O a values are given as 1 mole of residue; amounts less than 1 mole of 0,1 residue are indicated. not present. Residue numbers predicted for the ARH-m sequence are shown in parentheses. . Tryptic peptide composition is 200μ of individual column wax! From the amount of division It is; 200μ! The peptide peel is written at the end. A RH- m Cys residues are derived from the co-elution of disulfide-bonded peptides after tryptic digestion. It is inferred.

Example 5 Enzyme assay and enzyme activity A. Enzyme assay The activity against yeast tRNA (obtained from Calbiochem) was 18 Precipitation assay (5 hapi) with angiogenin or ARH-m at ~430 nM concentration ro et al., 1987, Proc, Natl, ^cad, Sci,, U, S,^1, Vol. 84, pp. 2238-2241). Figure 5a shows the activity of angiogenin and ARH-m measured against yeast tRNA . This assay tests the acid soluble fragments for 2 hours at pH 6, 8 and 37°C. measure the generation of Assays for pH profile were carried out at 37°C using the following buffers: Performed at pH value: 2-(N-morpholino)ethanesulfonic acid (Mes) , pH 5,4,5,8,6,3; N-(2-hydroxyethyl)piperazi: /-N'-2'-ethanesulfonic acid (Hepes) pH6.

2.6.8.7.3.7.8;) Lis(hydroxymethyl)aminoethane ( Tris), pH 7,5,8,1,8,5: and 2-(N-cyclohexylamide c) Ethanesulfonic acid (Ches) pH 8, 8, 9, 3 and 9.9゜pH value is Research Organics: C1e- veland, 0hio44125) using the pKa/℃ given by Calculated from readings at 25°C. Figure 5b shows ARH for yeast tRNA. -m (filled triangle), angiogenin (filled circle) and RNA a Figure 3 shows the effect of pH on the activity of the enzyme (filled squares). For comparison, Activity is normalized to the maximum ΔA obtained for each protein, 280.

The activity towards dinucleotide (3', 5') phosphate was reported by Shapiro et al. 988, ^na1. Biochem.

Volume 175, pp. 450-461): Halber and Vuarley (1989, Bi Oche 1stry, Vol. 28, pp. 1875-1884) Determined by HPLC quantitative method. The reaction mixture (30 μl) was 0.1 m Contains M substrate, 2.8aM ARH-In, and is incubated at 37°C for 4 to 24 hours. I held it.

The ribonucleolytic activity for 18S and 28S rRNA was determined using calf liver rRNA as a substrate. Using RNA (Pharacia), Shapiro et al. 25, pp. 3527-3532). did.

B. Enzyme activity ARH- for tRNA, 18S and 28S rRNA and dinucleotide substrates The enzymatic activity of m was very similar to that of angiogenin. t RNA assay In B, both proteins are related to the enzyme concentration at △A 260 to 1) H (37°C). showed a non-linear response, and its activity at the concentrations tested as shown in Figure 5a. not differentiated. ARH-m was somewhat different from angiogenin, but However, the maximum pH value was ~7.3 as shown in Figure 5b. Angioge Nin and RNNase available from CCaoper Biochemicals activity) has maximum activity under these assay conditions at pH ~6.8 and ~8.1; (1989b) by Lee and Vallee, respectively. , Biochem and Biophys.

Res, Commun, Vol. 161, pp. 121-126) match the results. Although angiogenin catalyzes the cleavage of dinucleotide substrates, This is approximately 5 to 6 times lower than that obtained for RNNase in It is known (5hapiro et al., 1988, An-al, Biochem, , Vol. 175, pp. 1450-461; Ha-rper and Vallee, 198 9, Biochemistry, Vol. 28, pp. 1875-1884). the Specificity is based on Kcatl Km values for four different dinucleotides. The specificity differs from that of the RNNase method. ARH-m for these substrates The activity of angiogenin was very similar to that of angiogenin, as shown in Table ■. in all cases , the KcatlKm value is 30% of the value obtained for angiogenin. and the specificity for the substrate remained unchanged.

Table n Activity of ARH-I [[1Kcat/Km (M -' S -') Substrate ARH-m Angiogenin RNase AeCp^ 13 12 6. OX 10’CpG 3.4 4.0 5. IX 10'U p＾　0.8　1.1　4. OX 106UpG O, 40, 41, 8X 10 'aARH-m and angiogenin assays: 30mM Mes, 30mM It was carried out in NaCl 1, 37°C and pH 6,0.

b Shapiro et al. (1988, ^na1. Biochem, Vol. 175, No. 4 (pp. 50-461) and Halper and Voualey (1989, Biochemis try, Vol. 28, pp. 1875-1884) Values from C. Halper and Vuerley (1989, supra); assays were performed at 25°C. The results were carried out spectrophotometrically in the buffer described above.

Angiogenin selectively cleaves naked 18S and 288 rRNA, resulting in significantly longer A product of approximately 100 to 500 nucleotides in length that remains resistant to further degradation for a long time is obtained. It is also known that (5 Hapiro et al., 1986, Bioch emistry, Vol. 25, pp. 3527-3532). such naked 18s The activity of AR) I-H against 28S rRNA and 28S rRNA is distinct from that of angiogenin. I couldn't tell. At time points taken over 60-90 minutes, two proteins ( 0.67 μM) was similar as revealed in agarose gel electrophoresis. Catalyzed the formation of products of size and strength.

Example 6 Suppression of protein synthesis and complete liposome cleavage in vitro The effect of ARH-II on protein translation in rabbit reticulocyte lysate was , a modification of the method of C1air et al. (1987, Proc. Natl. , ^cad, Sci, U, S, A.

, Vol. 84, pp. 8330-8334) by Halper and Vuerley. (1989, Biochemistry, Vol. 28, No. 187) 5-1884 pages) was measured. The reaction mixture contains angiogenin or ARH-1f[1 Contains 0-210 nM.

ARH-m catalyzed cleavage of reticulocyte liposomal RNA in intact liposomes is induced by St. Using the method of Kreyer et al. (1987, supra), Halper and Vuerley (19 89, supra).

Angiogenin has high specificity of 188 rRNA in 4OS liposome subunits. catalyzes the cleavage of molecules, St. Kreyer (1987, supra, 1988, Bioch. emistry, Vol. 27, pp. 7263-7268). in rabbit reticulocyte lysate by producing a small number of different products such as It is known to inhibit translation. On the other hand, many parts of RNNase method liposomes and form a large number of different sized reaction products. Angi of the two Ogenin is up to 1 million times faster than RNNase with other RNA substrates. Although slower, it is more effective in inactivating liposomes.

This remarkable specificity is only observed with intact liposomes or liposome subunits. or is not observed with isolated 18S and 28S rRNA.

As shown in Table 1, ARH-III inhibits protein synthesis in rabbit reticulocyte lysate. Significantly less effective than angiogenin in inhibiting. angiogenin completely inhibits protein translation at a concentration of 40-70 nM, whereas ARH-m Even a 3 times longer incubation time produces less than 50% inhibition at 210 nM. same A comparison of the proteins required to elicit the degree of repression indicates that ARH-m It is 20 to 30 times less active than angiogenin in the assay system. In vitro protein translation using ARH-Iff, angiogenin and RNNase methods Inhibition control sample concentration (nM) inhibition (%) A RH-III 70 0 10b44 Angiogenin 1015 RNNase method 30 30 a. Assays were performed in a rabbit reticulocyte lysate system. Suppression percentage Based on the amount of [358]-labeled protein recovered by acid precipitation: 0 % and 100% inhibition correspond to 6100Qcp■ and 200cpml, respectively. .

b The reaction time of ARH-H in the lysate mixture was 45 minutes instead of the usual 15 minutes. It was a minute.

Does ARH-m cleave liposomal RNA in the same selective manner as angiogenin? rRNA from the rabbit reticulocyte lysate reaction mixture to determine whether isolated, radiolabeled, and applied to a urea/polyacrylamide gel as shown in Figure 6. Analyzed by pneumophoresis.

In particular, rabbit reticulocyte lysate was treated with H20 for 15 minutes (lines 1 and 9), angiogel at 10 nM (line 2), at 3Q nM (line 3) and at 70 nM (line 4), or A RH-m constant at 30 nM (line 5), 70 nM (line 6), and 210 nM (line 7). Incubate and incubate at 210 nM for 45 minutes (line 8). Then rRNA [γ-32 P]-ATP and electrophoresed on a polyacrylamide gel. It was made visible by autoradiography. Samples in lines 1 to 4 in Figure 6 and electrophoresed for 5 minutes before feeding lines 5-9. ARH-m is essentially produce a cleavage product with the same pattern as angiogenin, but to do so requires more Required high concentration. Thus, with 20 nM angiogenin, the incubation time was ARH-11r210 to obtain product with the same strength as produced in 15 minutes Biological activity at 45 min incubation time at nM Angiogenesis is described by Knighton et al. (Kntghton et al., 1977, Br, J, C Ancer, Vol. 35, pp. 347-356) using the chicken embryo CAM assay. (Fett et al., 1985, Bioche@1stry, vol. 2) 4, pp. 5480-5486). individual app Sei used a set of 10 to 20 eggs. The total number of eggs used is ARH-If 24881 for angiogenin, 125 for angiogenin, and 2 for H,O There were 0 pieces. Angiogenin was also obtained from the E. coli expression system described in Example 3. and contained a -(-1) group: the effect is that the pin The effect was indistinguishable from that of natural angiogenin containing loglutamic acid. (5hapiro et al., 1988, Anal, Bfochet, No. 17 5, pp. 450-461). Dosage of ARH-III in CAM assay The response is different from angiogenin as shown in Figure 7. Typically, an Giogenin reaches maximum activity at a dose of approximately 1 ng, with positive activity in 50-60% of eggs. A response occurs. Its activity at 0.1 nf is 15-20% lower than that observed in water alone. Usually close to the background level of the ras.

In contrast, ARH-m has sufficient activity at 0.1 ng; was over 10 times more effective than angiogenin. RNase itself causes angiogenesis The ARH-m hybrid with inducing activity is characterized by the structure and function of anguisgenin. This region contains an RNase-inducing segment, which is recognized to be a That was not expected. At higher doses, ARH-Iff increased by 41% plus the maximum It elicited a large response, which was slightly smaller than the maximal response of angiogenin.

Example 8 Binding of ARH-Iff to PRI Resolve the apparent secondary association constant, Ka, for the binding of ARH-I[[ to PRI. Lee & Vallee, 1989a SBiochemi (1989a, B iochemistry, Vol. 28, pp. 219-224). To test the competition between ARE-1-111 and RNase for PRI, as It was decided by. PRI is Blackburn; 1979, J. Biol.

Chet, Vol. 254, pp. 12484-12487). Refined as such. Interaction (lie 0, IM Mes, 0.1M NaCL 1m MEDTA, recorded at pI-16,0 at 25°C. ARH-1f [to The Ka value obtained for this was the average of seven different determinations. ARH-m and P The Ka value determined for the association of RI is (4,3 ± 0.5) x 108 M-I It was S-1. This value is related to angiogenin as shown in Table ■ It was more than twice as large.

Dissociation rate constant, Kd Hariichi and Vouray (1989, supra) and Hariichi et al. 89b, Biochemistry.

28, pp. 225-230). protein as a function of time in the presence of an excess of scavenger. It was determined by measuring ARH-III release from the tar complex. Free Angi Ogenin and ARH-III were analyzed by cation exchange HPLC over a period of 10 days. was measured. Incubate at 25°C in the above buffer containing 120 μM DTT. carried out. The dissociation rate for the ARH-I[:PRI complex is shown in Table ■. The dissociation rate was substantially slower than that for angiogenin. Similarly, for 10 days protein:angiogenin and ARH- The Y-axis in Figure 8 showing the dissociation of m is unbound angiogenin or ARH-I. It shows the proportion of II. Approximately 8% of the angiogenin:PRI complex over 10 days dissociated, but there was no detectable dissociation of the ARH-1[[PRI complex (<2% ). Thus, the upper limit of Kd for ARH-I[[ is conservatively 3X10- It can be set to 8S-'.

Ki for the interaction of ARH-III and PRI is calculated from Ka and Kd values. It was ≦7×10-17M. This is angiogenin and RNNase at least 10 and 600 times lower than the values for

■Table Coupling constant 1 between ARH-m and PRI Trial fee Ka Kd Ki (If-IS-"xlO-8) (s-'x107) (fM) ARH-III 4.3 ≦0.3 <0.07 Angiogenin b1.8 1.3 0.7RN Aze A'') 3.4 150 40a Constant temperature maintenance conditions are 0.1MMes, 0.1 MNaCj, 1mM EDTAS pH 6,0, 25°C.

b IJ-et al. (1989b, Biochemistry, Vol. 28, No. 22 5-230).

In view of the foregoing, specific embodiments of the invention will now be described to provide a more detailed explanation. However, it is recognized that many modifications can be made without departing from the scope of the invention. will be valued. Accordingly, the invention is not limited except as by the claims.

Difference (chi) Sameness (chi) vul Fragment 7) A 7 Ragment Day Fragment Cvu 1 FIG.5 pH FIG.6 Positive response (%) O p dissociation (chi) Summary book Generating a DNA sequence encoding a mutant protein with increased angiogenesis-inducing activity Local mutagenesis of the gene for angiogenin to produce Ru. Expression vectors containing these sequences were introduced into host cells, and a clear increase in leading to the production of a mutant angiogenesis-inducing protein with angiogenesis-inducing activity. Ann other amino acids in the region of residues 8 to 22 of giogenin or the corresponding amino acids; In particular, the substitution of amino acids corresponding to residues 7 to 21 of RNase surprisingly Mutant angiogenesis with 10-fold increased ability to induce angiogenesis in allantoic assay Ogenin/RNase/hybrid protein was produced. In vivo and in vitro In the mutant hybrid protein, other angiogenin-related activities are altered. Book The mutant angiogenin/RNase hybrid protein according to the invention can be used in mammals. for promoting the development of the vascular network in or for the treatment of wounds, especially torn It is useful as a pharmaceutical composition for promoting the treatment of fibrocartilaginous or traumatic fibrocartilaginous lesions. .

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Claims (15)

[Claims] 1. in the region of angiogenin residues 8 to 22 or to angiogenin residues 8 to 22 Corresponding amino acids are replaced by other amino acids and increased angiogenesis Mutant angiogenin protein with inducing activity. 2. Region of angiogenin residues 8-22 or corresponding to angiogenin residues 8-22 according to claim 1, wherein the region of RNase is replaced with a region of amino acids in RNase. Mutant angiogenin protein. 3. The corresponding region of amino acids in RNase is the amino acid of RNase residues 7-21. The mutant according to claim 2, consisting of amino acids corresponding to acid or RNase residues 7 to 21. Mutant angiogenin protein. 4. An isolated and purified DNA sequence consisting of a DNA sequence encoding the protein of claim 1. 5. An isolated and purified DNA sequence consisting of a DNA sequence encoding the protein of claim 2. 6. An isolated and purified DNA sequence comprising a DNA sequence encoding the protein of claim 3. 7. An isolated vector for expressing the protein of claim 1 in transformed host cells. 8. An isolated vector for expressing the protein of claim 2 in transformed host cells. 9. An isolated vector for expressing the protein of claim 3 in a transformed host cell. 10. A host cell transformed with the vector of claim 7. 11. A host cell transformed with the vector of claim 8. 12. A host cell transformed with the vector of claim 9. 13. An angiogenesis-inducing effective amount of the protein of claim 1 in a pharmaceutically acceptable carrier. A pharmaceutical composition comprising: 14. An angiogenesis-inducing effective amount of the protein of claim 2 in a pharmaceutically acceptable carrier. A pharmaceutical composition comprising: 15. An angiogenesis-inducing effective amount of the protein of claim 3 in a pharmaceutically acceptable carrier. A pharmaceutical composition comprising: